Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC

Abstract

Atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) provides novel insight into dsRNA for RNAi applications.

Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores

“…Next, we optimized the mobile phase conditions for the ssRNAs separation. The separation patterns of low-range ssRNAs were compared using an octadecyl (C 18 )-based COSMOSIL RNA-RP1 column with super-wide pores using three different mobile phases consisting of mixtures of HFIP-TEA [ 23 , 24 ] and TEAA [ 25 , 26 ], which are commonly used for oligonucleotide separation. Since acetonitrile did not dissolve in the HFIP-containing mixture, when such a mixture was used as solvent A, only methanol was used as solvent B. Nucleic acids are easily adsorbed on various surfaces, so in HPLC analysis, when the peak of the target nucleic acid is not detected or the analysis data are not reproducible, the nucleic acid may have been adsorbed on the column or piping.…”

Strategies for the production of dsRNA biocontrols as alternatives to chemical pesticides

“…IVT reactions can also produce by-products comprised of short products formed by inefficient transcription, often referred to as "shortmers." Additionally long contaminants or "longmers" may arise, either from promoter-less transcription, extension by priming using RNA-dependant RNA synthesis (Gholamalipour et al, 2018(Gholamalipour et al, , 2019, or as a result of the formation of multimers or aggregates of dsRNA (Nwokeoji et al, 2019). However, short single-stranded overhangs are easily removed by incubation with a ssRNA-specific ribonucleases such as RNase T1, in order to produce blunt end fragments.…”

“…While deliberate utilisation of this mechanism in the dual polymerase T7-ϕ6 system produces effective insecticidal dsRNA, random primer extension results in double-stranded hairpin regions at the end of single stranded transcripts, which will subsequently affect the binding of complimentary ssRNA transcripts into dsRNA, and how key RNAi pathway enzymes such as Dicer effectively process these dsRNA substrates. IVT reactions can also produce by-products comprised of short products formed by inefficient transcription, often referred to as “shortmers.” Additionally long contaminants or “longmers” may arise, either from promoter-less transcription, extension by priming using RNA-dependant RNA synthesis ( Gholamalipour et al, 2018 , 2019 ), or as a result of the formation of multimers or aggregates of dsRNA ( Nwokeoji et al, 2019 ). However, short single-stranded overhangs are easily removed by incubation with a ssRNA-specific ribonucleases such as RNase T1, in order to produce blunt end fragments.…”

Analysis and Purification of Ssrna and Dsrna Molecules Using Asymmetrical Flow Field Flow Fractionation

“…3 -5 , Table 1 ). Weak intermolecular interactions can be reversed by moderate heat denaturation [53] . We compared migration of the heat-denatured and non-denatured pre-purified 300 nt ssRNA sample in agarose gel (Fig.…”